The development of gene therapy for inherited or acquired diseases is dependent on the establishment of safe and efficient gene delivery systems. One method for in vivo gene transfer uses viruses to transfer the exogenous gene into cells, but this method also transfers viral genes into the cells which may produce undesirable effects. In addition, contamination by wild type viruses is also a risk in this method.
Another method for in vivo gene transfer uses a complex of positively charged liposomes (composed of synthetic lipids) that bind to the DNA (the DNA is not encapsulated in the liposomes) (Zhu, N., et al., Science, 261:209-211, 1993). However, the toxicity of positively charged liposomes (Raz, E., et al., In: Vaccines, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y., 1994) limits the use of this method.
A complex of poly-L-lysine coupled to DNA and a ligand which can be targeted to the cell surface was also used for in vivo gene transfer (Wu, G. Y. and Wu, C. H., J. Biol. Chem., 263:14621-14624, 1988; Perales, J. C., et al., Proc. Natl. Acad. Sci., USA 91:4086-4090, 1994). However, transient expression of the transferred gene and immunogenicity of the complex limits the use of this method. Neutral or negatively charged liposomes (composed of synthetic or natural lipids) that encapsulate DNA were hitherto used for in vivo gene transfer (Nicolau, C., et al., Proc. Natl. Acad. Sci., USA, 80:1060-1072, 1983; Liebiger, B., et al., Biochem. Biophys. Res. Commun., 174:1223-1231, 1991).
Such liposomes are non-toxic, non-immunogenic and biode-gradable (Storm et al. In: Gregoriadis, G. (Ed), Liposome Technology, 2nd Ed. CRC Press, Baca Rator, Fla. 1993, 00.345-383) and are therefore good candidates for repetitive high dose treatment. However, the gene transfer efficiency in these experiments was low. One possible explanation for the low efficiency is that the DNA, which is encapsulated in liposomes and enters the cytoplasm through the lysosome (Cudd, A., and Nicolau, C., Biochim. Biophys. Acta, 845:477-491, 1985), is degraded by active DNases in the lysosome.
Therefore, treatments that inhibit lysosome activities can increase the amount of liposome-encapsulated DNA inside the cells.
Indeed, adenovirus particle and peptides derived from the N-terminal of the Influenza virus hemagglutinin subunit HA-2, which can disrupt membrane at low pH and therefore disrupt the lysosome membrane, were shown to enhance in vitro gene transfer efficiency when coupled to poly-L-lysine and DNA (Curiel, D. T., et al., Proc. Natl. Acad. Sci., USA, 88:8850-8854, 1993: Wagner, E., et al., Proc. Natl. Acad. Sci., USA, 89:7934-7938, 1992).
The efficiency of cationic-liposome mediated in vitro DNA transfection into cells was also shown to be increased when the cationic-liposomes were mixed with the DNA to be transfected together with two additional peptides derived from the Influenza virus hemagglutinin protein to form a complex between these three components (Kamata, H. et al. Nuclear Acid Research 22:536-537, 1994).
Fusion of liposomes prepared by reverse-phase evaporation (RPE) carrying DNA or protein molecules to target cells was shown to be mediated by Sendai virus proteins (Kameda, Y. et al, Exp. Cell Res. 173:56, 1987) or influenza virus proteins (Lapidot, M., Loyter, A., Exp. Cell Res. 189:241-246, 1990; Tikchonenko, T. I. et al, Gene 63:321-330, 1988) which were introduced into the liposome membrane (i,e, were present on the liposome's outer surface). The introduction of the DNA or protein encapsulated in the liposome was dependent on the presence of an active viral fusion protein and, therefore, either intact Sendai or influenza virus particles or their reconstituted envelopes were required. Liposomes containing modified reconstituted viral envelopes integrated in their lipid bilayer (Gould-Fogerite, S. et al, Gene, 84, 429-438, 1989) were also prepared by the protein-cochleate technique. Such liposomes were used for stable gene transfer and expression in animals.
All the above mentioned liposomes have large viral particles or reconstituted viral envelopes integrated in their lipid membrane which protrude outwards from the liposome membrane. In vivo administration of liposomes carrying such large viral particles may be hindered by the high antigenicity of such large particles as well as occasionally by their toxic effects.